Description: The recent discovery that bacteria and archaea employ an RNA-guided DNA cleavage mechanism to defend themselves from invasive genetic elements offers an unprecedented opportunity for understanding fundamental microbial biology and for developing biotechnology tools. Clustered, regularly interspaced, short palindromic repeats (CRISPR) loci encode three types of mechanistically different RNA-guided DNA cleavage enzymes that degrade invasive DNA while avoiding self-DNA. Understanding the molecular mechanisms of how these distinct DNA cleavage enzymes control their activities has important implications in basic enzymology, antibiotics resistance epidemics, human microbiome research, and genome editing. The Li laboratory has identified and purified representative members of two major types (Types II and III) of CRISPR-Cas DNA cleavage enzymes and is poised to unveil novel molecular mechanisms as well as to develop useful tools. Though both types are RNA-guided and invader-specific, these nucleases have drastically different in enzyme composition and activation mechanisms. An integrated approach ranging from cell-based assays, to structural biology and to fundamental enzymology will be employed to compare and contrast the mode of DNA interference by these nucleases, leading to an understanding of how microbe impact human health and biosphere and to an ultimate goal of developing CRISPR-based technology. The Li laboratory has assembled a team of scientists with complementary expertise in microbiology, nucleic acid biochemistry, mammalian cell biology, X-ray crystallography, and high-throughput cryogenic electron microscopy, in order to maximize the impact while mitigating risks of the research. Relevance: The CRISPR elements are found in more than 40% bacteria and are critical to maintenance of the overall microbial environment. The frequent occurrence of CRISPR in medically important bacteria that include but not limited to Yersinia pestis, Mycobacterium tuberculosis, Haemophilus influenzae, Helicobacter pylori, Neisseria meningitides, Vibrio vulnificus, Staphylococcus aureus, Salmonella Typhi, Clostridium tetani, and human microbiome relates CRISPR directly to human health. A thorough understanding of the CRISPR immunity has important implications in eradicating virulence and creating new antimicrobial strategies. While one of the CRISPR enzymes, namely Cas9, has been repurposed to serve as a user-specified genome-editing tool with ever-increasing popularity, we are yet to unleash the full potential of the CRISPR-derived tools in biomedical applications. The proposed research is aimed at overcoming current limitations while expanding the capability.